Fertilization in mammals begins with a unique cell-cell adhesion event involving the sperm protein IZUMO and the oocyte protein JUNO that enables interaction of fusigenic proteins to induce fusion of the sperm and egg plasma membranes. The currently accepted model implies that gamete fusion occurs spontaneously and that the oocyte is a passive participant. However, our recent finding that sperm-oocyte contact induced activation of the FAK-family member PYK2 in the oocyte cortex, and that PYK2 was required for sperm incorporation, showed that the oocyte is an active participant in the fertilization process. Our proposed hypothesis is that sperm-oocyte adhesion results in trans-interactions between surface proteins on the sperm and oocyte that lead to recruitment and activation of PYK2 within the adjacent oocyte cortex. PYK2 activity then promotes actin polymerization and microvillus elongation that increases the area of membrane contact allowing transient fusion pores to become permanent. PYK2 also controls remodeling of the cortical actin later to allow the sperm head to enter the cytoplasmic compartment of the oocyte. Subsequently, we propose that PYK2 activity spreads laterally through the oocyte cortex to initiate global remodeling of the cortical actin layer, which is essential for optimization of the Ca2+ signaling machinery in the oocyte. The proposed hypothesis is significant because it implies that `outside-in' signaling between sperm and oocyte plays an important role in zygote development that is bypassed during the Intracytoplasmic Sperm Injection (ICSI) where sperm-oocyte surface interactions do not occur. The objective of this proposal is to identify sperm and oocyte surface proteins that initiate PYK2 signaling, define the pathway by which PYK2 controls cytoskeletal remodeling and its impact on oocyte Ca2+ signaling machinery, and establish whether artificial activation of PYK2 might improve the outcome of ICSI using the mouse and bovine systems where ICSI is usually inefficient. Aim 1 will identify the sperm and oocyte surface proteins required to induce PYK2 activation at fertilization. A microsphere-based immobilized ligand system will be tested to develop an effective way of inducing oocyte PYK2 activation artificially. Aim 2 will elucidate the pathway by which PYK2 induces actin filament assembly and cell process elongation at the point of sperm-oocyte contact. This part of the study will identify specific proteins that could be targeted pharmacologically to enhance cortical actin remodeling in oocytes. The third aim will define the specific role that PYK2 plays in maintaining optimal Ca2+ signaling capability within the oocyte cortex. Experiments will then test whether artificial activation of PYK2 by microsphere-immobilized sperm proteins can improve the outcome of ICSI in mouse and bovine oocytes. Together, the hypothesis and goals presented in this proposal represent a significant step forward in our understanding of oocyte activation, and have the potential to greatly improve the efficiency of Assisted Reproductive Techniques used to improve fertility in domestic animals and humans.